(A) The four individual helices forming the core of the Lipocone superfamily are consistently colored across the illustrated representatives. The inter-helix linkers are colored gray, and lineage-specific synapomorphic insertions and extensions are colored light brown. Active site and other residues of interest are rendered as ball-and-stick. Protein Data Bank (PDB) IDs or Genbank accessions used to generate AF3 models are provided. (B) Relationship network of the Lipocone families. The thickness of the edges is scaled by the negative-log HHalign p-values. Families are colored according to the community identified by the Leiden algorithm (37) (see Methods). (C) Box plots displaying core helix transmembrane propensity scores of individual sequences within different Lipocone families. The horizontal divider represents the boundary between typical TM and soluble sequences.

Sequence logo of conserved core elements of the Lipocone families.

These correspond to the core helices H2, H3, and H4. The three conserved active site residue positions are boxed in dotted lines with the inferred ancestral residue indicated at the top of the alignment. Families are grouped and labeled on the left in their higher-order clades.

Known and predicted Lipocone reaction mechanisms.

Experimentally supported reactions are boxed in blue (A-B), while a predicted reaction based on genome displacement by a Lipocone domain of an experimentally characterized enzyme is boxed in orange (C). The remaining reactions (D-G) are suggested based on the contextual inferences in this work. Attacking and leaving groups are denoted by dashed green and red circles, respectively.

Reconstructed evolutionary scenario for the Lipocone superfamily.

The relative temporal epochs are demarcated by vertical lines and labeled at the bottom. The clades are represented by colored lines indicating the maximum depth to which the families listed to the right can be traced. Colors track the superkingdom-level phyletic distribution of the family. Dashed-line circles indicate uncertainty in the origin of lineage(s). Inferred or experimentally characterized functions for families are indicated to the left of family names. Asterisks denote newly described families.

Representative contexts for the Lipocone superfamily, grouped by shared functional themes.

Genes are depicted by box arrows, with the arrowhead indicating the 3’ end of genes. Genes encoding proteins with multiple domains are broken into labeled sections corresponding to them. Domain architectures are depicted by the individual domains represented by distinct shapes. TMs, lipoboxes (LPs), and SPs are depicted as unlabeled, narrow yellow, blue, and red rectangles, respectively. All Lipocone domains are consistently colored in orange. Genes marked with asterisks are labeled by the Genbank accession number below each context. Colored labels above genes denote well-known gene names or gene cluster modules. Abbreviations: PTase, peptidase; TFase, transferase; GlycosylTFase, Glycosyltransferase; MPTase, metallopeptidase; TGase, transglycosylase; SLP, serine-containing lipobox; cNMPBD, cNMP-binding domain; NCPBM, novel putative carbohydrate binding module; (w)HTH, (winged) helix-turn-helix; ZnR, Zinc ribbon; PPTs, pentapeptide repeats; Imm, immunity protein; βPs, β-propeller repeats; Cystatin-FD, Cystatin fold domain; MTase, methylase; PGBD, peptidoglycan-binding domain; MβL, metallo-β-lactamase; L12-ClpS, ClpS-ribosomal L7/L12 domain; TA, teichoic acid.

Lipocone contextual network.

The network represents the conserved contextual associations of Lipocone domains (hexagonal nodes). Nodes and edges are colored based on known or inferred functional categories of the domains. The nodes are scaled by their degree. Gray coloring indicates domains without specific functional assignments. Examples of conserved gene neighborhoods and domain architectures supplementing those in Figure 5 illustrate contexts that bridge functional themes. Here, individual domains are colored to match network coloring. Additional abbreviations to those in Figure 5: APH-Pkinase, aminoglycoside phosphotransferase-like kinase; HUP, HIGH, UspA and PP-ATPase superfamily-like domain; Alk-phosphatase, Alkaline phosphatase; dehyd, dehydrogenase; TPRs, tetratricopeptide repeats; PMM/PGM, phosphomannomutase/phosphoglucomutase; ZnF, zinc finger; APC-transporter, amino acid-polyamine-organocation transporter; LPS, lipopolysaccharide.